DESCRIPTION: (Applicant's Abstract) The broad, long-term objective of the proposed research is to develop an improved method for producing animal models of inherited retinal degenerations, so that the mechanisms by which specific mutations lead to photoreceptor cell death can be better understood, and rational therapies to prevent vision loss can be devised. While major progress had been made in identifying mutations that cause retinal degenerations in the past decade, the mechanisms by which these mutations lead to blindness are not completely understood. Naturally occurring animal models of retinal degeneration have been useful for the study of the pathogenesis, but the number of such models is limited. Engineered animal models of disease, such as transgenic and knockout mice, have also been used, but these methods suffer from several limitations. Expression of wild-type transgenes in the retina often leads to retinal degeneration. The phenotypes obtained from gene knockout techniques are not always the same as those produced by single base mutations. A technique for creating specific, single base changes in genomic DNA has recently been developed. This approach uses chimeric RNA-DNA oligonucleotides to induce single base pair changes into DNA, and has been called chimeroplasty. Successful use of chimeroplasty to produce single-nucleotide conversions in genomic DNA of cells in vitro and in vivo has been reported. The goal of the proposed research is to develop the use of chimeroplasty for creating animals with targeted mutations in photoreceptor genes, to use as models of retinal disease. The specific aims are to: 1. To develop and optimize chimeroplasty in mouse embryonic stem (ES) cells using reporter genes; 2. To determine if chimeroplasty can be used to introduce single base changes into the genomic DNA of mouse ES cells; and 3. To test the ability of mouse ES cells treated with chimeric oligonucleotides to generate chimeric mice and contribute to the germline. While the focus of this work is retinal degenerations, chimeroplasty could be used to generate mutations in any desired gene, and thus could be extended to study disease pathogenesis in general, and to investigation of protein function.